Project Summary/Abstract Early in embryonic development three facial prominences come together and fuse to form a continuous upper lip. This multistep process often fails, leading to cleft lip in ~1/700 newborn infants. We know that fusion will fail if the prominences do not grow towards each other, but it can also fail after the prominences touch if the surface cells fail to reach out to and then intermingle with the surface cells of the neighboring prominence. Apparently these dynamic behaviors of surface cells (reaching out and then intermingling) are tightly controlled, because we only see these behaviors where facial prominences should be fusing (e.g. to form the upper lip) and not elsewhere (e.g. the tongue to the roof of the mouth). However we know nothing about how these dynamic behaviors are regulated. We have recently used single cell RNA sequencing to learn the characteristics of the different cells that participate in fusion, including the transcription factors that give the cells their identity, the signaling proteins that permit them to communicate with each other and the cytoskeleton elements that direct their dynamic (or not so dynamic) behaviors. From these data we have discovered that a special population of surface (basal epithelial) cells are found only at the fusion zone. These cells have a unique signaling signature, as well as a unique program for cytoskeletal dynamics. We hypothesize that their signaling signature turns on dynamic behaviors of adjacent cells to initiate and then propagate fusion. As a corollary, we predict that modulating these signals should modulate the efficiency of fusion. To test these ideas we will develop methods for simultaneously visualizing signaling, cell identity and dynamic behaviors (cellular extensions) in fixed whole mouse faces during fusion. We will also develop methods for culturing faces during fusion so that we can study the process in real time, outside of the mother. Finally, we will manipulate signaling in these explants to ask whether we can modulate the efficiency of fusion. In this way, we expect to define the cell populations that participate in fusion, the genetic programs that direct their unique behaviors and the signaling interactions that coordinate and regulate fusion. If we are successful, we will identify signaling pathways that regulate the efficiency of fusion, pathways that could be developed as targets for therapeutic interventions to reduce the incidence of cleft lip in newborn infants.